Capillary array

ABSTRACT

A capillary array includes a light detection portion, a sample supply portion, a buffer solution supply portion and a voltage application portion which are necessary functions for electrophoresis, thereby, when assembling the capillary array into an electrophoresis apparatus, the same can be immediately used. Accordingly, a capillary array is provided which can be easily incorporated into an electrophoresis apparatus.

This is a continuation of application Ser. No. 11/300,377, filed Dec.15, 2005, now U.S. Pat. No. 7,785,458 which is a continuation ofapplication Ser. No. 09/845,303, filed May 1, 2001, now U.S. Pat. No.7,005,052, issued Feb. 28, 2006, which are hereby incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a capillary array used for a capillaryarray electrophoresis apparatus which separates and analyzes samplessuch as DNA and protein.

2. Conventional Art

An application technology in which an array is constituted by combininga plurality of capillaries, an electrophoresis medium and a sample to beseparated and analyzed are supplied to the respective capillaries andmoved therethrough to thereby separate and analyze the object sample iswell known, wherein a sample such as DNA and protein labelled by afluorescent material is supplied to the capillaries. Such applicationtechnology is, for example, disclosed in U.S. Pat. Nos. 5,366,608,5,529,679, 5,516,409, 5,730,850, 5,790,727, 5,582,705, 5,439,578 and5,274,240. In view of a through-put of the separation and analysis, itis much more advantageous to use electrophoresis with multi capillariesrather than electrophoresis with a flat plate gel.

JP-A-9-96623 (1997) discloses an application technology which separatesand analyzes a fluorescent labelled sample through electrophoresis bymaking use of a multi-capillaries.

A capillary array electrophoresis apparatus is basically constituted bysuch as a capillary array, an excitation light system including a laserbeam source, a light receiving optical system which detects fluorescenceand a voltage application unit which causes electrophoresis. In suchcapillary array electrophoresis apparatus the capillary array isconstituted by aligning a plurality of capillaries in a plane shape, anda laser beam is irradiated to the capillaries which are filled by asample (fluorescent sample) labelled by a fluorescent material inparallel direction with the capillary aligning direction, then, throughthe lens action of the capillaries the laser beam is condensed and thelaser beam is irradiated to the fluorescent sample in all of thecapillaries when the laser beam is irradiated, the fluorescent sampleemits fluorescence. Through detection by the light receiving opticalsystem of the fluorescence emitted from the fluorescent sample in adirection substantially perpendicular to the laser beam irradiationdirection, the measurement of the sample is performed.

The above patent document discloses a schematic diagram of a detectionportion for the array, but does not disclose an entire structure of aspecific capillary array for assembling the same into theelectrophoresis apparatus.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a capillary arrayhaving a specific structure which is suitable for a capillary arrayelectrophoresis.

The present invention is to provide a capillary array which comprises alight detection portion, a buffer solution injection portion and anelectrode built-in capillary head. The capillary array of the presentinvention is provided with functions necessary for an electrophoresisapparatus.

More specifically, the present invention provides a capillary arraywhich comprises a plurality of capillaries which includes a polymerprotective film on the surface thereof and of which one ends are bundledand of which other ends are spread; a light detection portion in whichthe capillaries are juxtaposed each other and are aligned substantiallyon a plane and the polymer protective films therein are removed; a headwhich holds the spread capillaries integrally, an electrode which isbuilt-in in the head, electrically connected to the head and is immersedin a sample solution; and another electrode provided at the bundledcapillaries.

Another embodiment of the present invention provides a capillary arrayin which one ends of a plurality of capillaries with a protective coatare bundled and the end portion thereof are aligned in flat so as toform a buffer solution injection port; the other ends of the capillariespenetrate through a capillary head with a built-in electrode and areinserted into metal tubes which are connected electrically to thebuilt-in electrode, a light detection portion is formed at anintermediate portion of the capillary array wherein the protective coatof the capillaries is removed, the protective coat removed capillariesare sandwiched between first and second support substrates, a windowwhich permits emission of fluorescence is formed on one of the first andsecond support substrates and a black coating is formed on the other ofthe first and second support substrates at a position corresponding tothe fluorescence emission permitting window on the one supportsubstrate.

One of the support substrates at the light detection portion of thecapillaries can be processed to provide a groove which permits laserbeam passage so as to reduce fluorescence reflection from the bottomthereof. The capillaries at the capillary head are cut in an alignment,are inserted closely into corresponding tubes and are secured thereto.One of the methods of securing is to injecting a gluing agent and tocure the same. Through attachment of a cap for protecting the sampleinjection port, transportation, handling and management of the capillaryarray can be performed safely. Further, a possible drying of an open endof the capillary array of which use is interrupted can be prevented. Theends of the capillaries at the sample injection port are slightlyprojected from the metallic tubes.

In the light detection portion, a reflection light shielding film isprovided at the opposite side of the window permitting passage offluorescence. At the sample supply portion for the capillary array ametallic tube electrode is provided which is electrically connected tothe electrode for the array head and into which the capillaries areinserted and are secured within the metallic tubes such as by a gluingagent.

The top end of the sample supply portion is adapted so as to permitattachment of a cap for containing buffer solution, thereby, the top endof the sample supply portion is protected during transportation thereof.When interrupting separation and analysis after assembling the capillaryarray into the electrophoresis apparatus and performing the separationand analysis, buffer solution is introduced into the cap to therebyprevent the top ends of the capillaries from drying, thus a conditionpermitting reuse of the capillaries at any time can be maintained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing an electrophoresis system to whicha capillary array of the present invention is applied;

FIG. 2 is a perspective view showing a structure of a capillary arrayaccording to the present invention;

FIG. 3 is an exploded view showing a structure of a light detectionportion for a capillary array according to the present invention;

FIG. 4 is a schematic cross sectional view of a structure of anon-irradiation portion in a light detection portion for a capillaryarray according to the present invention;

FIGS. 5A and 5B are views for explaining action of black coating in FIG.3;

FIG. 6 is a plane view of FIG. 3;

FIG. 7 is a perspective view partly being cut out for explaining astructure of and around a load header used in the present invention;

FIG. 8 is a cross sectional view for explaining a specific structure oftop ends of capillaries used in the present invention;

FIG. 9 is a perspective view for explaining a relationship between theload header and a protective cap used in the present invention; and

FIG. 10 is a perspective view showing a structure of a capillary headportion according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinbelow, embodiments of the present invention will be explained indetail with reference to the drawings.

FIG. 1 is a schematic diagram when a capillary array of the presentinvention is applied to an electrophoresis system. A plurality ofcapillaries, for example, 16 pieces of capillaries, are collected toform an array. At a light detection portion 29 a bottom support plate(glass substrate) “a” and a top support plate (silicon substrate) “b”are provided, and at a window portion thereof a transparent portion ofthe capillaries which is formed by removing the polyimide coat thereofis provided. FIG. 2 shows an entire structure of the capillary arrayaccording to the present invention which includes capillaries 1, thelight detection portion 29, a capillary head 30 and a load header 31with a built-in electrode. The top ends of the capillaries are insertedinto electrode tubes 32 and are secured thereto. A voltage forelectrophoresis is applied between the capillary head 30 and the loadheader 31.

In FIG. 1, the laser beam 33 generated from the laser beam source 20 isdivided into two parts by the beam splitter 22 and the advancingdirection thereof is change by the mirror 21. The laser beam 33 iscondensed by a condenser lens 23 and is irradiated to the capillaries 1from a direction in parallel with the alignment direction of thecapillaries 1. The inside of the capillaries 1 is filled with the samplelabelled by a fluorescent material (fluorescent sample 34), and when thelaser beam 33 is irradiated the fluorescent sample 34 emits fluorescence35. For the detection of the fluorescence 35, the fluorescence 35emitted in substantially perpendicular direction with respect to thealignment plane of the capillaries 1 is converted into parallel light bya first lens 24, is effected of image division by an optical filter andimage division prism 25, and thereafter image formed on the CCD camera27 by a second lens 26 and is detected by the CCD camera 27. Thedetected measurement data is processed by a processing unit 28.

In FIG. 1, the laser beam 33 is irradiated from the both sides of thelight detection portion 29, however, the apparatus can be constituted insuch a manner that the laser beam 33 is irradiated only from one sidethereof. Further, the layout of the light receiving optical system isnot limited to that illustrated in FIG. 1. Still further, the number ofconstituting capillaries 1 is not limited to 16 pieces and the structureof the buffer solution injection port 30 and the conductive fluorescentsample injection port 32 is not also limited to those shown in FIG. 1.

Now, an operation sequence of the capillary array electrophoresisapparatus will be explained. The buffer solution 36 contained in thebuffer solution container 17 is injected into the capillaries 1 from thebuffer solution injection port 30. Subsequently, the conductivefluorescent sample injection port 32 is immersed in the fluorescentsample container 18 filled with the fluorescent sample 34 and thefluorescent sample 34 is injected into the capillaries 1. Thereafter,the conductive fluorescent sample injection port 32 is immersed in abuffer solution container (not shown) containing a buffer solution, anda high voltage is applied between the buffer solution injection port 30and the fluorescent sample injection port 32 by the high voltage powersource 19 to thereby cause electrophoresis in the capillaries. Since themoving speed by electrophoresis is proportional to the electric chargemagnitude of the molecules and is reverse proportional to the mass ofthe molecules, the fluorescent sample 34 is separated. Throughcontinuous application of the high voltage for a long time theelectrophoresis is caused for a long time and the fluorescence 35emitted at this time is continuously measured.

A detailed structure of the light detection portion is shown in FIG. 3.As shown in FIG. 3, between a glass substrate 3 and a silicon substrate2 the capillaries 1 where a portion 9 being removed of polyimide coat 9is formed are sandwiched. On the glass substrate 3 a groove 4 is formedwhich permits passage of laser beam and the bottom of the groove 4 isfinished into ground glass. Further, other than the groove portion ofthe glass substrate 3 forms a laser beam non-irradiation portion 5.

The silicon substrate 2 is provide with a window frames 7 formingwindows 6 through which fluorescence is taken out. A black coating 46 isformed outside the glass plate 3 to thereby reduce noises caused byreflection of fluorescence.

FIG. 4 shows a cross sectional view of the laser beam non-irradiationportion 5 in FIG. 3. The surface of the glass substrate 3 where thepolyimide coating contacts is processed in such a high accuracy thatinterference fringes can be observed on the surface and the flatnessdegree thereof is high. A plurality of capillaries 1 are contacted tothe highly flattened surface via the polyimide coating 10 and arealigned thereon. Thereby, the plurality of the capillaries 1 follow theglass substrate 3 and are aligned thereon with high accuracy and easily.V shaped grooves 8 are formed on the silicon substrate 2 and thecapillaries 1 are aligned within the grooves 8.

FIGS. 5A and 5B show cross sectional views at the laser beam irradiationportion 4 in FIG. 3, wherein FIG. 5A is a view for explaining when noblack coating 46 is formed and FIG. 5B is a view for explaining when theblack coating 46 is formed.

When no black coating 46 is provided as shown in FIG. 5A, laser beam 47penetrates and passes through the plurality of capillaries which arealigned in a high accuracy at this moment, scattered light 51 from thesurface of the fused quartz tubes 9 passes through the glass substrate 3and is irradiated to a fluorescent emitting material on the surface ofthe opposing member 49 disposed opposite to the glass substrate 3, andthe fluorescence 52 emitted thereby returns to the quartz tubes 9,further passes the through window 6 and is directed to the first lens 24which causes noises. Further, when a fluorescent emitting material 50deposited on the back face of the glass substrate 3, such likely causesnoises.

However, when the black coating 46 is applied on the back face of theglass substrate 3 as shown in FIG. 5B, even if a fluorescent emittingmaterial 50 is contained in the opposing member 49 and further afluorescent emitting material 50 is deposited after the black coating 46is applied, the scattered light 51 is absorbed by the black coating 46,thereby, the causes of noises are removed. As a material of the blackcoating 46 a paint which emits no fluorescence is used. As a typicalpaint application work a silk screening is used, however, other paintingmethod can be used, and further a manual painting can also be used.

FIG. 6 shows schematically a typical plane view seen from the top inFIG. 3. The silicon substrate 2 is shown by two dots chain lines. Thethrough window 6 provided on the silicon substrate 2 is also shown bytwo dots chain lines. Further, FIG. 6 shows a state where a plurality ofcapillaries are aligned for a capillary array.

With FIG. 6 an alignment of the capillaries will be explained. From theportions of the capillaries corresponding to the light detection portionthe polyimide resin 10 coating the fused quartz tubes 9 is removed. Theremoval was conventionally performed, for example, in such a manner thatafter removing the polyimide coating by a predetermined size one by oneseparately, then the removed portions are arranged. However, when thepolyimide coating is removed one by one by a predetermined removingwidth, a processing error is caused and the removed width varies.Further, the arrangement is performed in such a manner that the removedportions, in particular, the boundaries (the boundary where thepolyimide resin 10 is cut out) align each other, however, such operationlikely causes error and takes time. Usually, a non alignment of theboundary portion can be immediately recognized. In the worst case, aremaining polyimide resin can be observed from the through window 6which causes great adverse effect to the detection.

Therefore, instead of the one by one coating removal, after arrangingthe plurality of capillaries when the polyimide coating is removedcollectively, the removed portions of polyimide resin 10 on theplurality of capillaries are neatly aligned. It is easily recognizedwhich aligning method is used when observing the alignment of theboundaries. The predetermined width and the predetermined position ofthe polyimide resin removed position can be freely changed inclusivelywith the plurality of the capillaries.

FIG. 7 is a view for explaining a load header portion representing oneembodiment of the present invention. The load header portion isconstituted by such as capillaries 211, electrode SUS pipes 212 servingas metallic tubes, a holder 214, a holder cover 215 and an electrode216. The inside the holder 214 the electrode plate 216 of phosphorbronze through which the SUS pipes 212 are passed and welded thereto isassembled. As shown in FIG. 7, the capillaries 211 pass throughrespective holes in the holder cover 215 and the SUS pipes 212 andproject at the opposite end face of the SUS pipes 212 by less than 1 mm.

The top ends of 16 pieces of capillaries at the side of the load headerinto which the capillary head is assembled are cut and aligned to alength of L+10 mm from the center of the fluorescent detection portion.Wherein L is the length of a capillary from the center of thefluorescent detection portion to the top end of the load header aftercompleting assembly of the header, and is required depending onseparation performance of a sequencer and electrophoresis time to have apredetermined length as, for example, being designed in the length of220 mm, 360 mm, 500 mm and 800 mm. Subsequently, the capillaries 211 areinserted through the holes of the load header cover 215 into thecorresponding SUS pipes 212. After adjusting the respective capillaries211 so as to project from the top ends of the SUS pipes 212 by 10 mm, agluing agent is injected into the holes of the load header 215 tothereby secure the respective capillaries to the load header cover 215.

Now, a processing of the top end portion of the load header will beexplained. FIG. 8 is an enlarged cross sectional view for explaining thesame. As shown in the drawing, a gluing agent 217 is injected betweenthe SUS pipe 212 and the capillary 211 to seal the gap therebetween.Thereafter, with a cutting device using a blade the top end of thecapillary 211 is cut to a projecting length of 0.5˜1.0 mm from the endface of the SUS pipe 212. With these operations, the length of therespective capillaries from the top end of the load header to thefluorescent detection portion can be aligned to a predetermined length,thereby, the electrophoresis time for the 16 pieces of the capillariescan be uniformalized. Further, the sealing structure of the gap betweenthe SUS pipes 212 and the capillaries 211 is an indispensable measure toprevent the sample solution from penetrating into the gap as well as toprevent a possible carry-over when performing measurement on othersamples.

Now, the length of the capillaries 211 from the top ends of the SUSpipes 212 is required more than 0.5 mm so as to form an optimum electricfield for introducing DNA molecules into the capillaries. On the otherhand, as shown in FIG. 8, when measuring a micro amount of sample in adegree of micro litter, it is necessary to limit the top end length ofthe capillaries 211 below 1.0 mm. Therefore, the length of 0.5˜1.0 mm isa proper length both for the gluing agent injection work to the top endof the SUS pipes and the cutting work of top ends of the capillaries.

Another top end configuration of the SUS pipe 212 for the load headerrepresenting another embodiment of the present invention will beexplained. Although the entire outlook structure thereof issubstantially the same as the above embodiment, a SUS pipe 212 having aconically spread shape at a top end at the side being incorporatedinside the holder is used. As a result, when inserting capillaries 211from the holes in the load header cover 215 into the SUS pipes 212, thecapillaries 211 can be easily inserted even if the centers of the bothare slightly offset which produces an advantage of enhancingworkability.

FIG. 9 is a perspective view showing a relationship between the sampleinjection portion of the capillary array and a protective cap thereforin the present invention. The sample injection portion at the top end ofthe load header 130 for the capillary array is designed to permitfitting into a cap 341. Thereby, the capillary array is protected duringtransportation thereof, moreover, when temporarily interrupting anoperation of an electrophoresis apparatus using such capillary array orwhen storing the capillary array after removing the same from theelectrophoresis apparatus for some reason, if a buffer solution isintroduced into the cap 341 and the capillary array is immersed in thebuffer solution, the capillary array can be protected. A flange portion134 for the cap 341 is provided for achieving close contact with theload header 130.

FIG. 10 is a diagram showing a relationship between the capillary headand the protective cap therefor in the present invention. The capillaryhead 110 is designed to permit fitting into a cap 111 of silicon rubber.Thereby, like the protective cap 341, the capillary array can beprotected during transportation thereof. Further, when storing thecapillary array after removing the same from the electrophoresisapparatus, a possible drying of the top end of the capillary head can beprevented.

Each of the capillaries 1 used in the capillary array as explained aboveis a fused quartz tube having inner diameter of 50±10 μm and outerdiameter of 340±20 μm. Since the fused quartz tube itself breaks veryeasily, a polyimide coating having thickness of 15±5 μm is applied onthe surface of the capillary. In view of limiting amount of fluorescentsample 34 it is desirable to reduce the inner diameter of the capillary,however, on the other hand in view of a concave lens effect due torefractive index difference between the fluorescent sample 34 and fusedquartz, the capillary having a too small inner diameter makes themeasurement difficult. Therefore, the inner diameter of 50˜100 μm ispreferable for the quartz tube. Further, in order to suppress the aboverefractive index difference it is preferable that the outer diameter ofthe fused quartz tube is small, however, a too small outer diametermakes assembling thereof difficult because of static electricity,therefore, the outer diameter of 250˜350 μm is preferable for the fusedquartz tube. The coating material for the capillary 1 is not limited tothe polyimide, a material having an equivalent electrical insulation andother properties as those of polyimide can be used.

As has been explained above, the capillary array of the presentinvention is provided with basic functions necessary for electrophoresisand includes in integration the light detection portion, the voltageapplication portion and the buffer solution gel supply portion which arenecessary for incorporating into the electrophoresis apparatus.

According to the present invention, a capillary array can be providedwhich is easy to handle, ensures a sufficient mechanical protection andfacilities attachment and detachment to a concerned electrophoresisapparatus.

1. A set comprising a capillary array and a cap that is permitted toattach said cap to said capillary array when said capillary array isremoved from an electrophoresis apparatus and is stored, wherein saidcapillary array is removably attached to the electrophoresis apparatusand includes a plurality of capillaries, each being provided with asample injection port and an electrophoresis medium injection port, aholder portion that spreads the sample injection ports of the pluralityof capillaries from one another and holds the same, a light detectionportion in which a portion of the plurality of capillaries are alignedto which excitation light is irradiated, and said cap includes acontainer portion that contains buffer solution and a close contactmaking portion that closely contacts the holder portion, wherein saidcap is structured to be able to accommodate the sample injection portsof the capillaries therein when the close contact making portion isclosely contacted to the holder portion, and wherein the close contactmaking portion is a flange portion extending outward from the upperportion of the container portion.
 2. The set of claim 1, furthercomprising another cap that is configured to be fitted to a capillaryhead formed by bundling the electrophoresis medium ports.